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Liver function: Postprandial urea nitrogen elevation and indocyanine green clearance in the dog
TOXICOLOGY
AND
liver
APPLIED
Function:
10,577-585
PHARMACOLOGY
Postprandial
lndocyanine
Urea
Green
(1967)
Nitrogen
Clearance
Elevation
in the Dog1
EUGENE E. VOCIN,~ HELEN R. !&EGGS, DELWIN AND PAUL A. MATTIS Merck
institute
for
Therapeutic Research, Merck & Co., West Received
Merck Point, February
and
Sharp 6 Pennsyluania
L. BOKELMAN,
Dohme 19486
Research
bbomtories,
6, 1967
Since the liver has been shown to be the sole site of urea formation (Krebs and Henseleit, 1932; Krebs, 1952), and since, in the presence of hepatic damage, there is a decreased capacity for the organism to convert amino acids to urea, it is possible that the postprandial elevation of urea nitrogen in the plasma after a standard test meal might be a useful index for assessing liver function. Therefore, it was the purpose of this study to compare the elevation of urea nitrogen and the plasma half-life ( T0.6) o f intravenously injected indocyanine green (ICG) with two methods currently used for evaluation of liver function, and to correlate these findings with histopathologic changes in the presence of two hepatotoxins producing damage by different mechanisms. METHODS
Beagle dogs of both sexes, 6-10 months of age, were individually housed, allowed water ad libitum, and fed for about 1 hour daily with a commercial laboratory chow, Chemical determinations for glucose, protein, albumin, urea nitrogen, alkaline phosphatase, glutamic-oxalacetic transaminase, and uric acid were performed by standard methods on sera obtained by venipuncture following an 18-hour fasting period. The level of urea nitrogen was determined following collection of sera at specified intervals after the animals consumed 440 g of an all-meat commercial dog food (test meal) containing not less than 13% protein. This meal was selected because the dogs consumed the entire portion within 5-10 minutes after its presentation. The half-life of ICC: was determined after the intravenous administration of 0.5 or 1.0 mg/kg of the dye as previously described (Vogin et al., 1965). Forty-eight hours after the urea nitrogen determinations were made, several dogs were anesthetized with pentobarbital sodium (32.5 mg/kg, iv), and a laparotomy was performed. Wedge-shaped liver biopsies were obtained under asep* A preliminary report of this work was presented at the Fiftieth Annual Meeting of the Federation of American Societies for Experimental Biology, Atlantic City, New Jersey, April 11-16, 1966. *Present address: Food and Drug Research Laboratories, Maurice Avenue at Fifty-eighth Street, Maspeth, New York.
577
578
EUGENE
E.
VOGIN
ET
AL.
tic conditions at weekly intervals from these dogs until the animals were sacrificed. Carbon tetrachloride (2 ml/kg in 2 ml/kg of mineral oil) was administered to 7 dogs by gavage for 2 successive days, and the animals were then observed for 4 weeks. Ethionine (25 mg/kg) was administered orally to 11 dogs once daily, 5 days/week, for 4 weeks. Postprandial urea nitrogen in serum was determined in each dog prior to treatment with the hepatotoxic agent, 24 hours after the second dose of carbon tetrachloride, or on the third day of dosing with ethionine, and again at 7-day intervals until the dogs were sacrificed. Liver biopsies were obtained weekly from 3 dogs that received carbon tetrachloride and 3 dogs that received ethionine, and the histomorphologic findings were correlated with changes found in urea nitrogen, serum glutamic-oxalacetic transaminase, alkaline phosphatase, and ICC. The dogs were sacrificed with an overdose of a barbiturate in the fifth week for postmortem examination, and the tissues selected for histologic examination were fixed in 10% formalin. Hematoxylinand eosin-stained paraffin sections were prepared from liver and kidney of the carbon tetrachloride-treated dogs, and from liver, kidney, and pancreas of the ethionine-treated animals. For the demonstration of lipid, Oil Red O-stained frozen sections were employed. Additional stains included Alcian blue-periodic acid-Schiff, and the Prussian blue reaction for iron. Statistical analysis of the data was performed according to methods outlined in Snedecor ( 1956). RESULTS
Urea Nitrogen Elevation
In the control phase of the experiment the urea nitrogen in blood of normal dogs was elevated 1.4 +- 0.8 (SD) mg/lOO ml above fasting levels within 30 minutes after the test meal was consumed. The peak elevation in urea nitrogen occurred 4 hours after the test meal when serum urea nitrogen was 13.7 r+ 2.0 mg/lOO ml above fasting levels. Samples obtained at the eighth hour did not differ signiilcantly from the 4-hour samples, but did exhibit a greater variation between animals (the rise in urea nitrogen was 14.4 -C 5.2 mg/lOO ml). By hour 24 the slight elevation in urea nitrogen was not significant. The reproducibility of this response is exemplified in Table 1. Postprandial elevations in urea nitrogen were determined in 5 dogs at a 7-day interval. NO significant differences were noted between the two sets of data. Subsequent studies in other beagles provided similar results. Analysis of the data in Table 1 indicated that the least variations in the postprandial elevation of urea nitrogen occurred during the first 4-hour sampling period. Thus, in subsequent experiments blood sampling was limited to this time period. Effect of Carbon Tetmchloride-Induced
Hepatotoxicity
The effects of carbon tetrachloride administration on the levels of urea nitrogen obtained following the test meal in two replicate studies are shown in Fig.
LIVER
FUNCTION
IN
TABLE POSTPRANDIAL
RISE
IN SERUM
UREA
Time after
NITROGEN
THE
579
DOG
1 IN NORMAL
BEAGLES
AFTER
A
TEST MEAL
Increase in serum urea nitrogen over fasting level5 (mg/lOO ml)
meal
Test
(hr)
0.5 1 2 4 8 24
(1 Fasting serum urea nitrogen b Mean Z& SD of 5 dogs. Test
16
1.4 f 0.9 5.4 f 1.1 11.1 i 2.7 14.5
f
16.3 2.5
f f
Test
aa
1.4 f
0.6
4.6
f
1.4
10.2
f
a.2
2.5
13.0
ZIT 1.5
6.7 1.4
13.4 3.3
f f
was 14.0 i 3.4 % was performed
mg/lOO 7 days
ml. after
Test
3.7 2.0
1.
1. The urea nitrogen elevation in serum was markedly decreased at the 2-hour and 4-hour time intervals, 24 hours after the final dosage of carbon tetrachloride (drug week I). During the second week after damage, postprandial urea nitrogen elevation increased markedly at all time intervals and in each dog compared to the control week. In the’ third and fourth weeks after carbon tetra-
zo-
-1 -2 -3
18 s E" z s0 E z Q &! 3 Z
DRUG WEEK CONTROL
r-:
161412IO 86-
0’
’
0.5
I
I
I
I .o
2.0
4.0
TIME (HOURS AFTER TEST MEAL) FIG. 1. Effect of carbon tetrachloride-induced liver injury on the concentration of urea nitrogen in the serum of dogs at intervals after a test meal. Each curve represents the same group of seven dogs, and each point on the curve is the mean of the determinations on the seven animals. Liver injury was induced by two successive daily oral dosages of 2 ml/kg of carbon tetrachloride. The control curve is based on pretreatment determinations. The determinations for “drug week 1” were performed 24 hours after the second dose of carbon tetrachloride, and the remaining sets of determinations were performed at successive ‘I-day intervab after the first.
580
EUGENE
E.
VOGIN
ET
AL.
chloride administration, the postprandial urea nitrogen elevation, though slightly increased, was not significantly different from the control. However, more variation between individual dogs was observed. The effect of carbon tetrachloride on the clinical chemistry of a fasted dog is detailed in Table 2. Little or no significant change was noted in the fasting serum glucose, protein, or urea nitrogen level. The low glucose value seen during drug week 1 in this animal may be related to the anorexia observed in these dogs during the 2 days preceding blood collection when carbon tetrachloride was being administered. Upon cessation of carbon tetrachloride administration the food consumption in these animals returned to pretest levels, and during weeks 2 through 4, the glucose values were found to be within normal limits. TABLE EFFECT
OF ORAL
ADMINISTRATION
ON SOME
SERUM
Determination Glucose (mg/lOO ml) Protein (g/100 ml) Albumin (g/100 ml) Albumin : globulin ratio Urea nitrogen (mg/lOO ml) Alkaline phosphatase (units)c Serum glutamic-oxalacetic transaminase (units)d Dric acid (mg/lOO ml)
2 OF CARBON
CHEMISTRY
Weeka -2
Weeks
102
98 6.55 2.70 0.70 15.0 12
6.36 3.13 0.97 12.4 10
14 0.33
Weekb
-1
11
0.32
TETRACHLORIDE OF A DOG
VALUES
Weekb
1
0
64 7.11 3.40 0.92 13.8 53 >1350
89 7.40 2.98 0.67 14.0
33 19
0.70
0.57
Weekb 3
Week’ 4
88 7.75 2.87 0.59 14.3 19
89 7.55 2.88 0.62
19 0.41
5 0.48
11.0 17
a These are pretreatment values. b Carbon tetrachloride was given as two successive daily oral dosages of 2 ml/kg. The determinations for week 1 were performed 24 hours after the second dose of carbon tetrachloride. The determinations for weeks 2, 3, and 4 were performed at successive weekly intervals after week 1. c King-Armstrong units. d Karmen units.
Both alkaline phosphatase and serum glutamic-oxalacetic transaminase were markedly increased as a result of carbon tetrachloride-induced hepatotoxicity in week 1. In the second week, only alkaline phosphatase remained above control values while serum glutamic-oxalacetic transaminase levels were within normal limits. The serum levels of both enzymes were within normal limits during the third and fourth weeks after carbon tetrachloride administration. The elevation in the T0.5 of ICG after carbon tetrachloride is summarized in Table 3. Within 24 hours after carbon tetrachloride, a marked rise in To.5 was seen in all dogs. During the second week, all dogs in the first study continued to exhibit an elevated T0.5, as did 2 of the 3 dogs in the second study. In the third dog in replicate 2, the T0.5 was only slightly elevated above the normal value. In the third and fourth weeks after carbon tetrachloride administration, the T0.5 was within the normal range in one animal within each group, but significantly elevated in all remaining dogs.
LIVER
FUNCTION
IN
THE
581
DOG
The results of the ICG studies, which indicated liver damage for as long as 4 weeks, correlated well with the biopsies performed on animals in replicate 2. Histomorphologic examination of the biopsies obtained 3 days after the final dosage of carbon tetrachloride revealed the following changes: disruption of the normal pattern of liver cords in the central lobular area, vacuolation, infiltration of inflammatory cells, necrosis, and fibrosis. The cells were reduced in size and closely packed. Examination of biopsies obtained during the next 3 weeks indicated that regeneration had occurred and only slight changes from the normal pattern were evident. The marked fatty metamorphosis seen during week 1 was not evident in week 2. The regenerative processes that occurred at this time interval were characterized as residual amounts of damage as evidenced by trace amounts of fat in the central areas. Subsequent biopsy specimens (weeks 3 and 4) showed little or no additional change; however, iron deposition was noted from the third week onward in these animals. TABLE EFFECT
OF ORAL OF
Serum
Replicatea 1 2
3
ADMINISTRATION OF CARBON TETRACHLORIDE INDOCYANINE GREEN INJECTED INTRAVENOUSLY half-life
Pretreatment values
Weeke 1
7.8 (6.5-8.7) 6.2 (5.1-7.0)
63.0 (25.7-137.0) 33.0 (21.544.0)
of indocyanine Week” 2 30.1 (10.5-83.9) 10.0 ( 7.4-12.7)
ON THE SERUM INTO DOGS green
HALF-LIFE
(min)b Weeke 3 11.1 (8.5-12.8) 9.9 (5.9-12.5)
Week= 4 9.8 (7.6-11.7) 10.8 (5.6-15.9)
a There were 4 dogs in replicate 1, and 3 dogs in replicate 2. Replicate 1 received 1 mg/kg of dye intravenously, and replicate 2 received 0.5 mg/kg. b The range is given in parentheses. c Carbon tetrachloride was given as two successive daily oral dosages of 2 ml/kg. The determinations for week 1 were performed 24 hours after the second dose of carbon tetrachloride. The determinations for weeks 2, 3, and 4 were performed at successive weekly intervals after week 1.
Effect
of Ethionine-Induced
Hepatotoxicity
The effects of daily administration of ethionine on the elevation of urea nitrogen levels after ingestion of the test meal in these replicate studies are shown in Fig. 2. No significant differences were found between replicates. During the first week of ethionine administration, the postprandial rise in urea nitrogen levels of serum exceeded that seen in the control period, but in the second week it was essentially the same as in the control phase. In the third and fourth weeks of drug administration, marked depression of urea nitrogen elevation was found, and this depression was evident as early as 1 hour after the test meal. Fasting levels of urea nitrogen or glucose were not significantly different from control levels. Total protein and albumin decreased during the fourth week, while serum glutamic-oxalacetic transaminase and alkaline phosphatase increased significantly. Typical serum chemistry values from one of the dogs re-
582
EUGENE
E. VOGIN
ET
AL.
DRUG WEEK
20.0 -
l -
CONTROL
HI
18.0 -
-2 -3 -4
a? 16.0 2
14.0 -
gE
12.0 -
s z 3
10.0 -
;
6.0 -
z
4.0 -
8.0 -
2.0 0’
I
’
0.5
I
J
1.0 2.0 TIME (HOURS AFTER TEST MEAL)
4.0
FIG. 2. Effect of ethionine-induced liver injury on the concentration of urea nitrogen in the serum of dogs at intervals after a test meal. Liver injury was induced by daily oral dosages of ethionine, 25 mg/kg, 5 days/week, for 4 weeks. The control curve is based on pretreatment determinations. The determinations for “drng week 1” were performed on the third day of dosing with ethionine, and the remaining sets of determinations were performed at successive 7-day intervals after the first. Each point on the curves represents the mean of the determinations on 8-11 animals.
TABLE EFFECT
OF ORAL
ADMINISTRATION
Determination Glucose (mg/lOO ml) Protein (g/100 ml) Albumin (g/100 ml) Albumin : globulin ratio Urea nitrogen (mg/lOO ml) Alkaline phosphatase (units)c Serum glutamic-oxalacetic transaminase (units)d Uric acid (mg/lOO ml)
Weeka
Week”
-2
-1
100
4 ON SOME
OF ETHIONINE
100
SERUM
Weekb 1 102
CHEMISTRY Weekb
VALUES Week6
2
96
OF A Doa Weekb
3
91
4
99
6.45 3.62
6.59 3.40
7.36
7.40
6.90
5.47
4.02
3.69
1.28
1.07
1.20 15.8
0.99 14.1
3.26 0.90
2.12 0.63
21.2
8
16
16
69
PO
14
32
57 0.53
14.5 7
14.7 8
16
14
0.25
0.29
0.39
0.35
0.62
9.4
a These are pretreatment values. b Ethionine was given as daily oral dosages of 25 mg/kg, 5 days/week, for 4 successive weeks. The determinations for week 1 were performed on the third day of dosing with ethionine. The determinations for weeks 2, 3, and 4 were performed at successive weekly intervals after week 1. c King-Armstrong units. d Karmen units.
LIVER
FUNCTION
IN
THE
583
DOG
ceiving ethionine are shown in Table 4. Uric acid was increased in all animals during the third and fourth weeks of ethionine administration. The elevation of the To.s of ICC for the 3 ethionine replicates is presented in Table 5. The average values obtained indicate that the T0.5 was increased during the third week in all replicates. However, several animals continued to exhibit half-lives within normal limits. By the fourth week, all dogs exhibited marked elevation in the TO.+ TABLE EFFECT
OF
ORAL
ADMINISTRATION GREEN
Serum
Replicatea 1
2
Pretreatment values 7.0
f
7.9
1.8
half-life
(4.Z.1)
of indocyanine
Week=
green
Week0 2
1 7.2fl.O
8.2
i
4.6
-
(5.9-9.8)
3
5
OF ETHIONINE ON TRE SERUM HALF-LIFE INJECTED INTRAVENOUSLY INTO Doos
(4.2-4.9)
7.4 (7.3-7.5)
6.4 (4.6-7.8)
OF INDOCYANINE
(min)”
Week” 3 1.2
11.6
* 10.8
Weeke 4 2.1
13.6
f
1.8
12.4
(7.4-15.7) 16.2
(11.5-13.3)
(11.6-19.1)
(17.2-23.7)
21.4
a There were 5 dogs in replicate 1, 3 dogs in replicate 2, and 3 dogs in replicate 3. Replicates 1 and 2 received 1 mg/kg of dye intravenously, and replicate 3 received 0.5 mg/kg. * The range is given in parentheses. c Ethionine was given as daily oral dosages of 25 mg/kg, 5 days/week, for 4 successive weeks. The determinations for week 1 were performed on the third day of dosing with ethionine. The determinations for weeks 2, 3, and 4 were performed at successive weekly intervals after week 1.
No significant histomorphologic changes were noted in liver biopsies obtained during the first and second weeks of ethionine administration. Examination of biopsies obtained during the third and fourth weeks revealed evidence of ethionine toxicity. Examination of a biopsy obtained during the fourth week revealed enlargement of hepatic cells, complete disorganization of hepatic cords, and vacuolation in the cytoplasm. In addition, bile duct proliferation and bile stasis were evident. A similar, though less extensive pattern of damage was seen in biopsies obtained in the third week. Diffuse fatty metamorphosis was found during the third and fourth weeks in these dogs. Iron deposition was also seen in the sections obtained after the fourth week. DISCUSSION
The results of the present study demonstrate that urea nitrogen levels of serum rise in response to protein intake in the beagle as well as in man (Addis et al., 1947) and the rat (Nitsan et al., 1966). The rise in urea nitrogen levels was reproducible both within the same animal and between animals when a standard meat diet was utilized as the source of protein. The data presented in Table 1 show that the most sign&ant and least variable elevations occurred between the l- and 4-hour sampling periods following the test meal. Urea nitrogen levels in blood samples obtained 8 hours after the test meal were equiva-
584
EUGENE
E.
VOGIN
ET
AL.
lent to those seen in the 4-hour samples, but exhibited a greater degree of individual variation. The present studies also demonstrate that either rapid (carbon tetrachloride) or slow (ethionine) induction of hepatic damage can be assessed by the measurement of the decrease in the postprandial rise of serum urea nitrogen. Marked atrophy of the pancreatic acinar cells was noted in ethioninetreated animals. Thus, impairment of nitrogen absorption, characteristic of pancreatic deficiency (Hoffman, 1964), may have contributed to the greater depression in postprandial urea nitrogen elevation exhibited by ethionine-treated dogs as compared with carbon tetrachloride-treated animals at the time histopathologic changes in the liver were most evident. The potentiation of the postprandial urea nitrogen elevation seen in the carbon tetrachloride-treated dogs during the second week may reflect increased enzyme synthesis occurring during the process of liver regeneration. In the analysis of serum obtained after carbon tetrachloride inhalation in rats, Dinman has observed 2 peaks of enzymatic activity. The first peak occurred B-12 hours after exposure of the animals and correlated with pathologic changes accompanying carbon tetrachloride-induced toxicity. The second peak, evident at 48 hours after exposure, may represent increased enzyme leakage from regenerating cells. This point awaits further clarification. TABLE COMPARISON
OF THE VARIOUS
6
TESTS FOR DETERMINING LIVER OF ETHIONINE ADMINISTRATIONS
a Ethionine weeks. b Incidence total number
was administered
as daily
is expressed as the number of animals tested.
DURING
THE THIRD
WEEK
Incidence of positive response@
Test Alkaline phosphatase Serum glutamic-oxalacetic Indocyanine green Urea nitrogen elevation
INJURY
4/11 6/11
transaminase
7/11 lo/l1
oral
dosages
of animals
of 25 mg/kg, showing
5 days/week,
a positive
for 4 successive
response,
divided
by the
Table 6 represents a comparison of the four methods used in this study for the detection of hepatotoxicity in the ethionine-treated dogs. During the third week, when histopathologic changes first became evident, the depression of postprandial urea nitrogen elevation appeared to be the most effective clinical diagnostic test. The elevation of serum glutamic-oxalacetic transaminase and the To.s of ICG were about equally diagnostic, but not as indicative of the onset of the hepatic damage as the urea nitrogen test, while alteration in alkaline phosphatase was the least sensitive method. The elevation of the T0,5 as a liver function test has been proposed by several investigators (Cornelius, 1963) and has been adequately demonstrated in these ’ Personal of Michigan
communication from Dr. B. D. Dinman, Medical Center, Ann Arbor, ‘Michigan.
Institute
of Industrial
Health,
University
LIVER
FUNCTION
IN
THE
585
DOG
studies. Of particular interest to us was the clinical correlation between the elevated T0.5 and histologic evidence of residual liver damage in the carbon tetrachloride-treated animals. Of the four clinical methods employed in this study, the ICG test was the only method which provided evidence of lingering hepatic damage 3-4 weeks after exposure to the toxin. Unpublished observations from this laboratory have shown that the determination of the T0.5 of ICG was superior to measurement of the 30-minute bromsulfalein retention (5 “g/kg, iv) in the detection of residual hepatic damage after arsenic ingestion. In this instance the dogs had been in our colony for at least 6 months after their exposure to arsenic, and the clinical finding of an elevated To.5 of ICC was con&med as being due to hepatic damage. Thus, it would appear that the To.5 determination may prove to be more satisfactory for evaluating liver function in recovery studies than presently employed methods, while the onset of liver damage might be indicated earlier by postprandial elevation of urea nitrogen. Further work is necessary to confirm these inferences. SUMMARY A method for evaluation of liver function, based on the postprandial elevation of urea nitrogen in the serum of beagles is described. Biopsy specimens were obtained and alkaline phosphatase, serum glutamic-oxalacetic transaminase, and indocyanine green clearance also were determined after the induction of liver injury by carbon tetrachloride or ethionine. The postprandial elevation of urea nitrogen was significantly reduced by both agents. In ethioninetreated animals the depression of postprandial urea nitrogen elevation was the most effective of the clinical diagnostic tests indicating the onset of hepatotoxicity. Indocyanine green clearance, however, was the most satisfactory test for determining residual hepatic damage during the recovery studies after carbon tetrachloride administration. ACKNOWLEDGMENTS
Mr.
The authors wish to thank Mrs. C. ‘Mitchell, Mrs. J. Rosenberger, W. Scott for their assistance in performing these studies.
Mr.
J.
Boyd,
and
REFERENCES ADDIS, T., B-q E., Poo, L. J., and Yunv, D. W. (1947). The relationship between the serum urea concentration and the protein consumption of normal individuals. .I. Clin. lnuest. 26, 369-374. CORNELIUS, C. ( 1963). Liver Function in Clinical Biochemistry of Domestic Animals (C. E. Cornelius and J. J. Kaneko, eds.), pp. 225-301. Academic Press, New York. HOFFMAN, W. S. (1984). The Biochemistry of Clinical Medicine, 3rd ed., pp. 740-741. Year Book Medical Publishers, Chicago, Illinois. Kmms, H. A. ( 1952). Urea synthesis. In: The Enzymes (J. B. Sumner and K. Myrback, eds.), Vol. II, pp. 866-885. Academic Press, New York. Kmms, H. A., and HENSELEIT, K. ( 1932). Untersuchungen iiber die Hamstoffbildung im Tierkorper. Z. Physiol. Chem. 210, 33-66. NITSAN, Z., BOLOURCHI, S., and MICKELSEN, 0. (1966). Blood urea levels as influenced by the dietary protein. Federation Proc. 25, 299. SNEDECOR, G. W. (1956). Statistical Methods, 5th ed. Iowa State College Press, Ames, Iowa. VOGIN, E. E., Scoa-r, W., and MAT-US, P. A. (1965). Hepatic clearance of indocyanine green in the beagle. Proc. Sot. Exptl. Biol. Med. 119, 570573.
AND
liver
APPLIED
Function:
10,577-585
PHARMACOLOGY
Postprandial
lndocyanine
Urea
Green
(1967)
Nitrogen
Clearance
Elevation
in the Dog1
EUGENE E. VOCIN,~ HELEN R. !&EGGS, DELWIN AND PAUL A. MATTIS Merck
institute
for
Therapeutic Research, Merck & Co., West Received
Merck Point, February
and
Sharp 6 Pennsyluania
L. BOKELMAN,
Dohme 19486
Research
bbomtories,
6, 1967
Since the liver has been shown to be the sole site of urea formation (Krebs and Henseleit, 1932; Krebs, 1952), and since, in the presence of hepatic damage, there is a decreased capacity for the organism to convert amino acids to urea, it is possible that the postprandial elevation of urea nitrogen in the plasma after a standard test meal might be a useful index for assessing liver function. Therefore, it was the purpose of this study to compare the elevation of urea nitrogen and the plasma half-life ( T0.6) o f intravenously injected indocyanine green (ICG) with two methods currently used for evaluation of liver function, and to correlate these findings with histopathologic changes in the presence of two hepatotoxins producing damage by different mechanisms. METHODS
Beagle dogs of both sexes, 6-10 months of age, were individually housed, allowed water ad libitum, and fed for about 1 hour daily with a commercial laboratory chow, Chemical determinations for glucose, protein, albumin, urea nitrogen, alkaline phosphatase, glutamic-oxalacetic transaminase, and uric acid were performed by standard methods on sera obtained by venipuncture following an 18-hour fasting period. The level of urea nitrogen was determined following collection of sera at specified intervals after the animals consumed 440 g of an all-meat commercial dog food (test meal) containing not less than 13% protein. This meal was selected because the dogs consumed the entire portion within 5-10 minutes after its presentation. The half-life of ICC: was determined after the intravenous administration of 0.5 or 1.0 mg/kg of the dye as previously described (Vogin et al., 1965). Forty-eight hours after the urea nitrogen determinations were made, several dogs were anesthetized with pentobarbital sodium (32.5 mg/kg, iv), and a laparotomy was performed. Wedge-shaped liver biopsies were obtained under asep* A preliminary report of this work was presented at the Fiftieth Annual Meeting of the Federation of American Societies for Experimental Biology, Atlantic City, New Jersey, April 11-16, 1966. *Present address: Food and Drug Research Laboratories, Maurice Avenue at Fifty-eighth Street, Maspeth, New York.
577
578
EUGENE
E.
VOGIN
ET
AL.
tic conditions at weekly intervals from these dogs until the animals were sacrificed. Carbon tetrachloride (2 ml/kg in 2 ml/kg of mineral oil) was administered to 7 dogs by gavage for 2 successive days, and the animals were then observed for 4 weeks. Ethionine (25 mg/kg) was administered orally to 11 dogs once daily, 5 days/week, for 4 weeks. Postprandial urea nitrogen in serum was determined in each dog prior to treatment with the hepatotoxic agent, 24 hours after the second dose of carbon tetrachloride, or on the third day of dosing with ethionine, and again at 7-day intervals until the dogs were sacrificed. Liver biopsies were obtained weekly from 3 dogs that received carbon tetrachloride and 3 dogs that received ethionine, and the histomorphologic findings were correlated with changes found in urea nitrogen, serum glutamic-oxalacetic transaminase, alkaline phosphatase, and ICC. The dogs were sacrificed with an overdose of a barbiturate in the fifth week for postmortem examination, and the tissues selected for histologic examination were fixed in 10% formalin. Hematoxylinand eosin-stained paraffin sections were prepared from liver and kidney of the carbon tetrachloride-treated dogs, and from liver, kidney, and pancreas of the ethionine-treated animals. For the demonstration of lipid, Oil Red O-stained frozen sections were employed. Additional stains included Alcian blue-periodic acid-Schiff, and the Prussian blue reaction for iron. Statistical analysis of the data was performed according to methods outlined in Snedecor ( 1956). RESULTS
Urea Nitrogen Elevation
In the control phase of the experiment the urea nitrogen in blood of normal dogs was elevated 1.4 +- 0.8 (SD) mg/lOO ml above fasting levels within 30 minutes after the test meal was consumed. The peak elevation in urea nitrogen occurred 4 hours after the test meal when serum urea nitrogen was 13.7 r+ 2.0 mg/lOO ml above fasting levels. Samples obtained at the eighth hour did not differ signiilcantly from the 4-hour samples, but did exhibit a greater variation between animals (the rise in urea nitrogen was 14.4 -C 5.2 mg/lOO ml). By hour 24 the slight elevation in urea nitrogen was not significant. The reproducibility of this response is exemplified in Table 1. Postprandial elevations in urea nitrogen were determined in 5 dogs at a 7-day interval. NO significant differences were noted between the two sets of data. Subsequent studies in other beagles provided similar results. Analysis of the data in Table 1 indicated that the least variations in the postprandial elevation of urea nitrogen occurred during the first 4-hour sampling period. Thus, in subsequent experiments blood sampling was limited to this time period. Effect of Carbon Tetmchloride-Induced
Hepatotoxicity
The effects of carbon tetrachloride administration on the levels of urea nitrogen obtained following the test meal in two replicate studies are shown in Fig.
LIVER
FUNCTION
IN
TABLE POSTPRANDIAL
RISE
IN SERUM
UREA
Time after
NITROGEN
THE
579
DOG
1 IN NORMAL
BEAGLES
AFTER
A
TEST MEAL
Increase in serum urea nitrogen over fasting level5 (mg/lOO ml)
meal
Test
(hr)
0.5 1 2 4 8 24
(1 Fasting serum urea nitrogen b Mean Z& SD of 5 dogs. Test
16
1.4 f 0.9 5.4 f 1.1 11.1 i 2.7 14.5
f
16.3 2.5
f f
Test
aa
1.4 f
0.6
4.6
f
1.4
10.2
f
a.2
2.5
13.0
ZIT 1.5
6.7 1.4
13.4 3.3
f f
was 14.0 i 3.4 % was performed
mg/lOO 7 days
ml. after
Test
3.7 2.0
1.
1. The urea nitrogen elevation in serum was markedly decreased at the 2-hour and 4-hour time intervals, 24 hours after the final dosage of carbon tetrachloride (drug week I). During the second week after damage, postprandial urea nitrogen elevation increased markedly at all time intervals and in each dog compared to the control week. In the’ third and fourth weeks after carbon tetra-
zo-
-1 -2 -3
18 s E" z s0 E z Q &! 3 Z
DRUG WEEK CONTROL
r-:
161412IO 86-
0’
’
0.5
I
I
I
I .o
2.0
4.0
TIME (HOURS AFTER TEST MEAL) FIG. 1. Effect of carbon tetrachloride-induced liver injury on the concentration of urea nitrogen in the serum of dogs at intervals after a test meal. Each curve represents the same group of seven dogs, and each point on the curve is the mean of the determinations on the seven animals. Liver injury was induced by two successive daily oral dosages of 2 ml/kg of carbon tetrachloride. The control curve is based on pretreatment determinations. The determinations for “drug week 1” were performed 24 hours after the second dose of carbon tetrachloride, and the remaining sets of determinations were performed at successive ‘I-day intervab after the first.
580
EUGENE
E.
VOGIN
ET
AL.
chloride administration, the postprandial urea nitrogen elevation, though slightly increased, was not significantly different from the control. However, more variation between individual dogs was observed. The effect of carbon tetrachloride on the clinical chemistry of a fasted dog is detailed in Table 2. Little or no significant change was noted in the fasting serum glucose, protein, or urea nitrogen level. The low glucose value seen during drug week 1 in this animal may be related to the anorexia observed in these dogs during the 2 days preceding blood collection when carbon tetrachloride was being administered. Upon cessation of carbon tetrachloride administration the food consumption in these animals returned to pretest levels, and during weeks 2 through 4, the glucose values were found to be within normal limits. TABLE EFFECT
OF ORAL
ADMINISTRATION
ON SOME
SERUM
Determination Glucose (mg/lOO ml) Protein (g/100 ml) Albumin (g/100 ml) Albumin : globulin ratio Urea nitrogen (mg/lOO ml) Alkaline phosphatase (units)c Serum glutamic-oxalacetic transaminase (units)d Dric acid (mg/lOO ml)
2 OF CARBON
CHEMISTRY
Weeka -2
Weeks
102
98 6.55 2.70 0.70 15.0 12
6.36 3.13 0.97 12.4 10
14 0.33
Weekb
-1
11
0.32
TETRACHLORIDE OF A DOG
VALUES
Weekb
1
0
64 7.11 3.40 0.92 13.8 53 >1350
89 7.40 2.98 0.67 14.0
33 19
0.70
0.57
Weekb 3
Week’ 4
88 7.75 2.87 0.59 14.3 19
89 7.55 2.88 0.62
19 0.41
5 0.48
11.0 17
a These are pretreatment values. b Carbon tetrachloride was given as two successive daily oral dosages of 2 ml/kg. The determinations for week 1 were performed 24 hours after the second dose of carbon tetrachloride. The determinations for weeks 2, 3, and 4 were performed at successive weekly intervals after week 1. c King-Armstrong units. d Karmen units.
Both alkaline phosphatase and serum glutamic-oxalacetic transaminase were markedly increased as a result of carbon tetrachloride-induced hepatotoxicity in week 1. In the second week, only alkaline phosphatase remained above control values while serum glutamic-oxalacetic transaminase levels were within normal limits. The serum levels of both enzymes were within normal limits during the third and fourth weeks after carbon tetrachloride administration. The elevation in the T0.5 of ICG after carbon tetrachloride is summarized in Table 3. Within 24 hours after carbon tetrachloride, a marked rise in To.5 was seen in all dogs. During the second week, all dogs in the first study continued to exhibit an elevated T0.5, as did 2 of the 3 dogs in the second study. In the third dog in replicate 2, the T0.5 was only slightly elevated above the normal value. In the third and fourth weeks after carbon tetrachloride administration, the T0.5 was within the normal range in one animal within each group, but significantly elevated in all remaining dogs.
LIVER
FUNCTION
IN
THE
581
DOG
The results of the ICG studies, which indicated liver damage for as long as 4 weeks, correlated well with the biopsies performed on animals in replicate 2. Histomorphologic examination of the biopsies obtained 3 days after the final dosage of carbon tetrachloride revealed the following changes: disruption of the normal pattern of liver cords in the central lobular area, vacuolation, infiltration of inflammatory cells, necrosis, and fibrosis. The cells were reduced in size and closely packed. Examination of biopsies obtained during the next 3 weeks indicated that regeneration had occurred and only slight changes from the normal pattern were evident. The marked fatty metamorphosis seen during week 1 was not evident in week 2. The regenerative processes that occurred at this time interval were characterized as residual amounts of damage as evidenced by trace amounts of fat in the central areas. Subsequent biopsy specimens (weeks 3 and 4) showed little or no additional change; however, iron deposition was noted from the third week onward in these animals. TABLE EFFECT
OF ORAL OF
Serum
Replicatea 1 2
3
ADMINISTRATION OF CARBON TETRACHLORIDE INDOCYANINE GREEN INJECTED INTRAVENOUSLY half-life
Pretreatment values
Weeke 1
7.8 (6.5-8.7) 6.2 (5.1-7.0)
63.0 (25.7-137.0) 33.0 (21.544.0)
of indocyanine Week” 2 30.1 (10.5-83.9) 10.0 ( 7.4-12.7)
ON THE SERUM INTO DOGS green
HALF-LIFE
(min)b Weeke 3 11.1 (8.5-12.8) 9.9 (5.9-12.5)
Week= 4 9.8 (7.6-11.7) 10.8 (5.6-15.9)
a There were 4 dogs in replicate 1, and 3 dogs in replicate 2. Replicate 1 received 1 mg/kg of dye intravenously, and replicate 2 received 0.5 mg/kg. b The range is given in parentheses. c Carbon tetrachloride was given as two successive daily oral dosages of 2 ml/kg. The determinations for week 1 were performed 24 hours after the second dose of carbon tetrachloride. The determinations for weeks 2, 3, and 4 were performed at successive weekly intervals after week 1.
Effect
of Ethionine-Induced
Hepatotoxicity
The effects of daily administration of ethionine on the elevation of urea nitrogen levels after ingestion of the test meal in these replicate studies are shown in Fig. 2. No significant differences were found between replicates. During the first week of ethionine administration, the postprandial rise in urea nitrogen levels of serum exceeded that seen in the control period, but in the second week it was essentially the same as in the control phase. In the third and fourth weeks of drug administration, marked depression of urea nitrogen elevation was found, and this depression was evident as early as 1 hour after the test meal. Fasting levels of urea nitrogen or glucose were not significantly different from control levels. Total protein and albumin decreased during the fourth week, while serum glutamic-oxalacetic transaminase and alkaline phosphatase increased significantly. Typical serum chemistry values from one of the dogs re-
582
EUGENE
E. VOGIN
ET
AL.
DRUG WEEK
20.0 -
l -
CONTROL
HI
18.0 -
-2 -3 -4
a? 16.0 2
14.0 -
gE
12.0 -
s z 3
10.0 -
;
6.0 -
z
4.0 -
8.0 -
2.0 0’
I
’
0.5
I
J
1.0 2.0 TIME (HOURS AFTER TEST MEAL)
4.0
FIG. 2. Effect of ethionine-induced liver injury on the concentration of urea nitrogen in the serum of dogs at intervals after a test meal. Liver injury was induced by daily oral dosages of ethionine, 25 mg/kg, 5 days/week, for 4 weeks. The control curve is based on pretreatment determinations. The determinations for “drng week 1” were performed on the third day of dosing with ethionine, and the remaining sets of determinations were performed at successive 7-day intervals after the first. Each point on the curves represents the mean of the determinations on 8-11 animals.
TABLE EFFECT
OF ORAL
ADMINISTRATION
Determination Glucose (mg/lOO ml) Protein (g/100 ml) Albumin (g/100 ml) Albumin : globulin ratio Urea nitrogen (mg/lOO ml) Alkaline phosphatase (units)c Serum glutamic-oxalacetic transaminase (units)d Uric acid (mg/lOO ml)
Weeka
Week”
-2
-1
100
4 ON SOME
OF ETHIONINE
100
SERUM
Weekb 1 102
CHEMISTRY Weekb
VALUES Week6
2
96
OF A Doa Weekb
3
91
4
99
6.45 3.62
6.59 3.40
7.36
7.40
6.90
5.47
4.02
3.69
1.28
1.07
1.20 15.8
0.99 14.1
3.26 0.90
2.12 0.63
21.2
8
16
16
69
PO
14
32
57 0.53
14.5 7
14.7 8
16
14
0.25
0.29
0.39
0.35
0.62
9.4
a These are pretreatment values. b Ethionine was given as daily oral dosages of 25 mg/kg, 5 days/week, for 4 successive weeks. The determinations for week 1 were performed on the third day of dosing with ethionine. The determinations for weeks 2, 3, and 4 were performed at successive weekly intervals after week 1. c King-Armstrong units. d Karmen units.
LIVER
FUNCTION
IN
THE
583
DOG
ceiving ethionine are shown in Table 4. Uric acid was increased in all animals during the third and fourth weeks of ethionine administration. The elevation of the To.s of ICC for the 3 ethionine replicates is presented in Table 5. The average values obtained indicate that the T0.5 was increased during the third week in all replicates. However, several animals continued to exhibit half-lives within normal limits. By the fourth week, all dogs exhibited marked elevation in the TO.+ TABLE EFFECT
OF
ORAL
ADMINISTRATION GREEN
Serum
Replicatea 1
2
Pretreatment values 7.0
f
7.9
1.8
half-life
(4.Z.1)
of indocyanine
Week=
green
Week0 2
1 7.2fl.O
8.2
i
4.6
-
(5.9-9.8)
3
5
OF ETHIONINE ON TRE SERUM HALF-LIFE INJECTED INTRAVENOUSLY INTO Doos
(4.2-4.9)
7.4 (7.3-7.5)
6.4 (4.6-7.8)
OF INDOCYANINE
(min)”
Week” 3 1.2
11.6
* 10.8
Weeke 4 2.1
13.6
f
1.8
12.4
(7.4-15.7) 16.2
(11.5-13.3)
(11.6-19.1)
(17.2-23.7)
21.4
a There were 5 dogs in replicate 1, 3 dogs in replicate 2, and 3 dogs in replicate 3. Replicates 1 and 2 received 1 mg/kg of dye intravenously, and replicate 3 received 0.5 mg/kg. * The range is given in parentheses. c Ethionine was given as daily oral dosages of 25 mg/kg, 5 days/week, for 4 successive weeks. The determinations for week 1 were performed on the third day of dosing with ethionine. The determinations for weeks 2, 3, and 4 were performed at successive weekly intervals after week 1.
No significant histomorphologic changes were noted in liver biopsies obtained during the first and second weeks of ethionine administration. Examination of biopsies obtained during the third and fourth weeks revealed evidence of ethionine toxicity. Examination of a biopsy obtained during the fourth week revealed enlargement of hepatic cells, complete disorganization of hepatic cords, and vacuolation in the cytoplasm. In addition, bile duct proliferation and bile stasis were evident. A similar, though less extensive pattern of damage was seen in biopsies obtained in the third week. Diffuse fatty metamorphosis was found during the third and fourth weeks in these dogs. Iron deposition was also seen in the sections obtained after the fourth week. DISCUSSION
The results of the present study demonstrate that urea nitrogen levels of serum rise in response to protein intake in the beagle as well as in man (Addis et al., 1947) and the rat (Nitsan et al., 1966). The rise in urea nitrogen levels was reproducible both within the same animal and between animals when a standard meat diet was utilized as the source of protein. The data presented in Table 1 show that the most sign&ant and least variable elevations occurred between the l- and 4-hour sampling periods following the test meal. Urea nitrogen levels in blood samples obtained 8 hours after the test meal were equiva-
584
EUGENE
E.
VOGIN
ET
AL.
lent to those seen in the 4-hour samples, but exhibited a greater degree of individual variation. The present studies also demonstrate that either rapid (carbon tetrachloride) or slow (ethionine) induction of hepatic damage can be assessed by the measurement of the decrease in the postprandial rise of serum urea nitrogen. Marked atrophy of the pancreatic acinar cells was noted in ethioninetreated animals. Thus, impairment of nitrogen absorption, characteristic of pancreatic deficiency (Hoffman, 1964), may have contributed to the greater depression in postprandial urea nitrogen elevation exhibited by ethionine-treated dogs as compared with carbon tetrachloride-treated animals at the time histopathologic changes in the liver were most evident. The potentiation of the postprandial urea nitrogen elevation seen in the carbon tetrachloride-treated dogs during the second week may reflect increased enzyme synthesis occurring during the process of liver regeneration. In the analysis of serum obtained after carbon tetrachloride inhalation in rats, Dinman has observed 2 peaks of enzymatic activity. The first peak occurred B-12 hours after exposure of the animals and correlated with pathologic changes accompanying carbon tetrachloride-induced toxicity. The second peak, evident at 48 hours after exposure, may represent increased enzyme leakage from regenerating cells. This point awaits further clarification. TABLE COMPARISON
OF THE VARIOUS
6
TESTS FOR DETERMINING LIVER OF ETHIONINE ADMINISTRATIONS
a Ethionine weeks. b Incidence total number
was administered
as daily
is expressed as the number of animals tested.
DURING
THE THIRD
WEEK
Incidence of positive response@
Test Alkaline phosphatase Serum glutamic-oxalacetic Indocyanine green Urea nitrogen elevation
INJURY
4/11 6/11
transaminase
7/11 lo/l1
oral
dosages
of animals
of 25 mg/kg, showing
5 days/week,
a positive
for 4 successive
response,
divided
by the
Table 6 represents a comparison of the four methods used in this study for the detection of hepatotoxicity in the ethionine-treated dogs. During the third week, when histopathologic changes first became evident, the depression of postprandial urea nitrogen elevation appeared to be the most effective clinical diagnostic test. The elevation of serum glutamic-oxalacetic transaminase and the To.s of ICG were about equally diagnostic, but not as indicative of the onset of the hepatic damage as the urea nitrogen test, while alteration in alkaline phosphatase was the least sensitive method. The elevation of the T0,5 as a liver function test has been proposed by several investigators (Cornelius, 1963) and has been adequately demonstrated in these ’ Personal of Michigan
communication from Dr. B. D. Dinman, Medical Center, Ann Arbor, ‘Michigan.
Institute
of Industrial
Health,
University
LIVER
FUNCTION
IN
THE
585
DOG
studies. Of particular interest to us was the clinical correlation between the elevated T0.5 and histologic evidence of residual liver damage in the carbon tetrachloride-treated animals. Of the four clinical methods employed in this study, the ICG test was the only method which provided evidence of lingering hepatic damage 3-4 weeks after exposure to the toxin. Unpublished observations from this laboratory have shown that the determination of the T0.5 of ICG was superior to measurement of the 30-minute bromsulfalein retention (5 “g/kg, iv) in the detection of residual hepatic damage after arsenic ingestion. In this instance the dogs had been in our colony for at least 6 months after their exposure to arsenic, and the clinical finding of an elevated To.5 of ICC was con&med as being due to hepatic damage. Thus, it would appear that the To.5 determination may prove to be more satisfactory for evaluating liver function in recovery studies than presently employed methods, while the onset of liver damage might be indicated earlier by postprandial elevation of urea nitrogen. Further work is necessary to confirm these inferences. SUMMARY A method for evaluation of liver function, based on the postprandial elevation of urea nitrogen in the serum of beagles is described. Biopsy specimens were obtained and alkaline phosphatase, serum glutamic-oxalacetic transaminase, and indocyanine green clearance also were determined after the induction of liver injury by carbon tetrachloride or ethionine. The postprandial elevation of urea nitrogen was significantly reduced by both agents. In ethioninetreated animals the depression of postprandial urea nitrogen elevation was the most effective of the clinical diagnostic tests indicating the onset of hepatotoxicity. Indocyanine green clearance, however, was the most satisfactory test for determining residual hepatic damage during the recovery studies after carbon tetrachloride administration. ACKNOWLEDGMENTS
Mr.
The authors wish to thank Mrs. C. ‘Mitchell, Mrs. J. Rosenberger, W. Scott for their assistance in performing these studies.
Mr.
J.
Boyd,
and
REFERENCES ADDIS, T., B-q E., Poo, L. J., and Yunv, D. W. (1947). The relationship between the serum urea concentration and the protein consumption of normal individuals. .I. Clin. lnuest. 26, 369-374. CORNELIUS, C. ( 1963). Liver Function in Clinical Biochemistry of Domestic Animals (C. E. Cornelius and J. J. Kaneko, eds.), pp. 225-301. Academic Press, New York. HOFFMAN, W. S. (1984). The Biochemistry of Clinical Medicine, 3rd ed., pp. 740-741. Year Book Medical Publishers, Chicago, Illinois. Kmms, H. A. ( 1952). Urea synthesis. In: The Enzymes (J. B. Sumner and K. Myrback, eds.), Vol. II, pp. 866-885. Academic Press, New York. Kmms, H. A., and HENSELEIT, K. ( 1932). Untersuchungen iiber die Hamstoffbildung im Tierkorper. Z. Physiol. Chem. 210, 33-66. NITSAN, Z., BOLOURCHI, S., and MICKELSEN, 0. (1966). Blood urea levels as influenced by the dietary protein. Federation Proc. 25, 299. SNEDECOR, G. W. (1956). Statistical Methods, 5th ed. Iowa State College Press, Ames, Iowa. VOGIN, E. E., Scoa-r, W., and MAT-US, P. A. (1965). Hepatic clearance of indocyanine green in the beagle. Proc. Sot. Exptl. Biol. Med. 119, 570573.